CN105722838B - The kinases γ of phosphatidylinositols 3 selective depressant - Google Patents

Abstract

The present invention relates to the compound of the selective depressant as PI3K γ.The present invention also provides the pharmaceutically acceptable composition comprising the compound and the method that various diseases, illness or obstacle are treated using said composition.

Description

Phosphatidyl-inositol 3-kinase-γ selective depressant

Technical field

The present invention relates to the compound of the inhibitor of the γ isoforms as phosphatidyl-inositol 3-kinase (PI3K γ).This hair It is bright that the pharmaceutically acceptable composition comprising the compound is also provided and the compound and the various obstacles of composition treatment are used Method.

Background technology

PI3K is the phosphorylation for the membrane lipid phosphatidylinositols (PI) being catalyzed on 3 '-OH of inositol ring to produce PI 3- phosphorus Sour [PI (3) P, PIP], PI 3,4- diphosphonic acid [PI (3,4) P₂, PIP2] and the triphosphoric acids of PI 3,4,5- [PI (3,4,5) P₃, PIP3] lipid kinase family.PI (3,4) P₂With PI (3,4,5) P₃It is used as the supplement site of various intracellular signalling proteins, its turn And Signaling complex is formed, so that extracellular signal to be transferred to the cytoplasmic surface of plasma membrane.

So far, it has been identified that the PI3K of 8 kinds of mammals, including four kinds of I classes PI3K.Ia classes include PI3K α, PI3K β and PI3K δ.All Ia fermentoids are all heterodimer compounds, and it includes the p85 aptamer subunits with the domain containing SH2 Related catalytic subunit (p110 α, p110 β or p110 δ).Ia classes PI3K is activated by tyrosine kinase signal transmission, and It is related to cell propagation and survives.PI3K α and PI3K β are also relevant with the tumour generation in various human cancers.Thus, PI3K α and PI3K β Pharmacological inhibitors are useful for treating various types of cancers.

PI3K γ are unique Ib classes PI3K members, are made up of catalytic subunit p110 γ, and p110 γ and p101 adjusts subunit It is related.PI3K γ are adjusted by G-protein coupled receptor (GPCR) via being combined with the β γ subunits of miscellaneous tripolymer G-protein Control.PI3K γ are main to express in hematopoietic cell and cardiac muscle cell, and is related to inflammation and mast cell function.Thus, PI3K γ Pharmacological inhibitors are useful for treating various inflammatory diseases, allergic reaction and angiocardiopathy.

Although many PI3K gamma inhibitors have researched and developed success, still need other compound to suppress PI3K γ, for treating various obstacles and disease.What is be especially desired to is with improved internal pharmacokinetics/pharmacodynamics behavior Those PI3K gamma inhibitors, for example, increase medicine exposed to target tissue, while those PI3K that non-targeted effect is minimized Gamma inhibitors.The larger exposure of per unit dose is exposed relative on target tissue to reduce exposure of missing the target.Generally, dose limitation Toxicity occur involving medicine is removed from circulation or for orally administering activating agent in the intestines and stomach (GI) removing device In official.Remove to reduce and improve the C added in blood plasma with bioavilability_max, while limit elimination organ (such as kidney, liver and GI the C in)_max.Reduce (improved bioavilability) in addition, absorbing increase and removing and typically result between patient in exposure side The variability in face is reduced, and thus also improves the security of the activating agent of administration.In addition.Show improved physical characteristic (for example Higher water solubility) activating agent be also desired.

The content of the invention

It has been found that compound (R) -6- (1- (fluoro ethyls of 2,2- bis-) -1H- pyrazoles -4- bases) -4,7,7- three of the present invention Methyl -2- (5- (the fluoro- l- hydroxyethyls of 2,2,2- tri-) pyridine 3- yls) -6,7- dihydro -5H- pyrrolo-es [3,4-b] pyridine -5- ketone And its pharmaceutically acceptable composition is effective and selective PI3K γ inhibitor, suppress when with other PI3K γ When agent is compared, it has improved pharmacokinetics/pharmacodynamic profiles.Therefore, it is a feature of the present invention that with following structure Compound：

Present invention also offers the compound comprising Formulas I and pharmaceutically acceptable carrier, auxiliary material or excipient (vehicle) pharmaceutical composition.These compounds and pharmaceutical composition are tight for treating various diseases or the mitigation disease Principal characteristic be it is useful, the disease include inflammatory disease and autoimmunity insufficiency of accommodation, for example asthma, atopic dermatitis, rhinitis, Anaphylactia, chronic obstructive pulmonary disease (COPD), septic shock, idiopathic pulmonary fibrosis, apoplexy, burn, arthropathy, class Rheumatic arthritis, systemic loupus erythematosus, atherosclerosis, acute pancreatitis, psoriasis, inflammatory bowel disease (IBD), ulcer Property colitis, Crohn's disease and Graves disease.

Research of the compound and composition that the present invention is provided for PI3K in biology and pathological phenomena, by these The kinase mediated research of intracellular signal transduction passage and the comparative evaluation of Azaindole kinase inhibitors is also useful.

Brief description of the drawings

Fig. 1 is shown with 2.5mg/kg BID, 5mg/kg BID and 10mg/kg BID (20mg/kg/ days) in treatment mouse glue The result for the compound 1 tested in arthritis (CIA) model that former albumen induces.

Fig. 2 is shown with 2.5mg/kg BID, 5mg/kg BID and 10mg/kg BID (20mg/kg/ days) in CIA models The result of the compound 1 of test.The result of those pawls of clinical arthritis sign is shown when being merely displayed in registration.

Fig. 3 is shown with 2.5mg/kg BID, 5mg/kg BID and 10mg/kg BID (20mg/kg/ days) in CIA models The result of the compound 1 of test.The result of those pawls without clinical arthritis sign is shown when being merely displayed in registration.

Fig. 4 is shown with the result of 5mg/kg BID and 10mg/kg the BID compounds 1 tested in IBD models.

Detailed description of the invention

Definition and general terms

Unless otherwise indicated, following definition used herein should be used.For purposes of the present invention, the mirror of chemical element Not according to the periodic table of elements, CAS editions, and Handbook of Chemistry and Physics, the 75th edition, 1994 are carried out.Separately Outside, the General Principle of organic chemistry is referring to " Organic Chemistry ", Thomas Sorrell, University Science Books, Sausalito：1999 and " March ' s Advanced Organic Chemistry ", the 5th edition, Smith, M.B. and March, J. are edited, John Wiley＆Sons, New York：2001, entire contents are incorporated by reference into Herein.

The compound for being decorated with the stereochemical center of determination is that spatial chemistry is pure, but with still undetermined absolute Spatial chemistry.Such compound can have R or S configurations.In those situations of wherein such absolute ownership after measured Under, chiral centre will be marked as R or S in drawing.Although compound 1 herein is drawn as R- configurations, this hair The embodiment of the racemic mixture of bright embodiment and R and S configurations including S- configurations.

The present invention is further characterized in that any compound comprising the present invention and pharmaceutically acceptable carrier, auxiliary material or tax The pharmaceutical composition of shape agent.

In one embodiment, it is a feature of the present invention that suppress patient PI3K kinase activities method, by The patient applies compound 1 or its pharmaceutical composition to carry out.In further embodiment, relative to PI3K- α, PI3K- β or PI3K- δ selective depression PI3K- γ.

In another embodiment, it is a feature of the present invention that treatment patient's is selected from following inflammatory and immunological regulation The disease or illness of sexual dysfunction or the method for mitigating its seriousness：For example, asthma, atopic dermatitis, rhinitis, anaphylactia, Chronic obstructive pulmonary disease (COPD), septic shock, idiopathic pulmonary fibrosis, apoplexy, burn, arthropathy, rheumatoid joint Inflammation, systemic loupus erythematosus, atherosclerosis, acute pancreatitis, psoriasis, inflammatory bowel disease, ulcerative colitis, Crow grace Family name's disease and Graves disease, are carried out by applying compound 1 or its pharmaceutical composition to the patient.

The present invention is further characterized in that the non-therapeutic method for suppressing PI3K- γ kinase activities in external biological sample, bag Including makes the biological sample contact compound 1 or the composition comprising the compound.

Composition, preparation and the administration of the present invention

In another aspect, the invention provides a kind of pharmaceutical composition of inclusion compound 1.For example, the present invention is provided A kind of inclusion compound 1 or its pharmaceutically acceptable derivates and pharmaceutically acceptable carrier, auxiliary material or excipient Pharmaceutical composition.In one embodiment, the amount of compound is to make it possible to effectively measurably press down in the present composition The amount of biological sample processed or the PI3K γ in patient.In one embodiment, the composition of the present invention is prepared, for needs The administration of the patient of said composition.In further embodiment, the composition of the present invention is prepared, is suffered from for oral give Person.Term " patient " used herein refers to animal, preferably mammal, optimal to choose.

It can also be appreciated that some the compounds of this invention can in a free form in the presence of and be used to treat, Huo Zhe In the case of suitable, exist as its pharmaceutically acceptable derivates.According to the present invention, pharmaceutically acceptable derivates bag Include but be not limited to pharmaceutically acceptable pro-drug, salt, ester, the salt of this kind of ester or any other adduct or derivative, Compound described elsewhere herein that once it can be directly or indirectly provided the patients of needs administration or its metabolin Or residue.Term " metabolin or residue of its inhibitory activity " used herein refers to that its metabolin or residue are also PI3K gamma inhibitors.

Term " pharmaceutically acceptable salt " used herein refers to such salt, in rational medical judgment scope, It is applied to the tissue of contact people and lower animal, without excessive toxicity, excitant, allergic reaction etc..

Pharmaceutically acceptable salt is well known in the art.For example, S.M.Berge et al. is in J.Pharmaceutical Sciences, 66：1-19, is described in detail pharmaceutically acceptable salt in 1977, is incorporated by reference into herein.Chemical combination of the present invention The pharmaceutically acceptable salt of thing is included from suitable inorganic and organic acid and salt derived from alkali.Pharmaceutically acceptable nontoxicity The salt of sour addition is the salt of the amino with inorganic acid or organic acid formation, the inorganic acid such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulphur Acid and perchloric acid, the organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, butanedioic acid or malonic acid, or pass through Use the salt of other methods used in the art such as amino of ion exchange formation.Other pharmaceutically acceptable salt bags Include adipate, alginates, ascorbate, aspartate, benzene sulfonate, benzoate, disulfate, borate, fourth Hydrochlorate, camphor hydrochlorate, camsilate, citrate, cyclopentane propionate, digluconate, lauryl sulfate, second sulphur Hydrochlorate, formates, fumarate, gluceptate, glycerophosphate, gluconate, Hemisulphate, enanthate, caproate, hydrogen Iodate, 2- isethionates, Lactobionate, lactate, laruate, lauryl sulfate, malate, maleic acid Salt, malonate, mesylate, 2- naphthalene sulfonates, nicotinate, nitrate, oleate, oxalates, palmitate, pamoate, Pectate, persulfate, 3- phenylpropionic acid salt, phosphate, picrate, Pivalate, propionate, stearate, amber Hydrochlorate, sulfate, tartrate, rhodanate, tosilate, hendecane hydrochlorate, valerate etc..Produced by appropriate alkali Salt include alkali metal, alkaline-earth metal, ammonium and N⁺(C_1-4Alkyl)₄Salt.The present invention also includes compound as disclosed herein Any Basic nitrogen-containing groups quaternized products.Water can be obtained by this kind of quaternization or oil is solvable or dispersible Product.Representational alkali metal or alkali salt include sodium, lithium, potassium, calcium, magnesium etc..If appropriate, others can pharmaceutically connect The salt received is using counter ion counterionsl gegenions such as halogen ion, hydroxyl, carboxylate radical, sulfate radical, phosphate radical, nitrate anion, C_1-8Sulfonate radical With the nontoxic ammonium salt of arylsulphonate formation, quaternary ammonium salt and amine cation salt.

As described above, the pharmaceutically acceptable composition of the present invention additionally comprises pharmaceutically acceptable carrier, auxiliary material Or excipient, carrier used herein, auxiliary material, excipient includes being suitable for the arbitrary and all molten of desired particular dosage form Agent, diluent or other liquid excipients, dispersing aid or suspending agent, surfactant, isotonic agent, thickener or emulsifying agent, Preservative, solid binder, lubricant etc..In Remington:The Science and Practice Pharmacy, the 21st Version, 2005, ed.D.B.Troy, Lippincott Williams＆Wilkins, Philadelphia, and Encyclopedia Pharmaceutical Technology, eds.J.Swarbrick and J.C.Boylan, 1988-1999, Marcel In Dekker, New York, various carriers for preparing pharmaceutically acceptable composition are disclosed and for its preparation Known technology, their content is all incorporated by reference into herein.(for example produce and appoint except incompatible with the compound of the present invention Why not good biological effect or other any other components with harmful way and pharmaceutically acceptable composition it is mutual Effect) any conventional carrier medium, it is using all covering within the scope of the invention.

It can include but is not limited to as some examples of the material of pharmaceutically acceptable carrier：Ion-exchanger, oxygen Change aluminium, aluminum stearate, lecithin, haemocyanin (such as human serum albumins), buffer substance (such as phosphate), glycine, Sorbic acid or potassium sorbate, the partial glyceride mixture of saturated vegetable fatty acid, water, salt or electrolyte (such as sulfuric acid milt egg In vain, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salt), cataloid, magnesium trisilicate, polyvinylpyrrolidone, poly- third Olefin(e) acid ester, wax class, polyethylene-polyoxypropylene block copolymer, lanolin, carbohydrate (such as lactose, dextrose and saccharose), starch (such as cornstarch and farina), such as cellulose and its derivates, sodium carboxymethylcellulose, ethyl cellulose and vinegar Acid cellulose；Powdered tragacanth；Malt；Gelatin；Talcum powder；Excipient such as cocoa butter and suppository wax；Oils such as peanut Oil, cottonseed oil；Safflower oil；Sesame oil；Olive oil；Corn oil and soybean oil；Glycol such as propane diols or polyethylene glycol；Esters, Such as ethyl oleate and ethyl laurate；Agar；Buffer, such as magnesium hydroxide and aluminium hydroxide；Alginic acid；It is pyrogen-free Water；Isotonic saline solution；Ringer's solution；Ethanol；And phosphate buffer, and other nontoxic compatible lubricants, such as bay Base sodium sulphate and magnesium stearate；According to the judgement of formulation scientist, there can also be colouring agent in the composition, releasing agent is coated Agent, sweetener, flavor enhancement and aromatic, preservative and antioxidant.

The composition of the present invention can be parenteral by oral administration, is sprayed by induction type, local, rectum, nose, cheek, intravaginal or Applied by implant bank.Term used herein is " parenteral " to include subcutaneous, intravenous, intramuscular, intra-articular, synovial membrane It is interior, intrathecal in breastbone, intraocular, in liver, internal wound, Epidural cavity, in backbone and intracranial injection or infusion techniques.Preferably, will Said composition by oral administration, intraperitoneal or intravenous administration.The Sterile injectable forms of the present composition can be aqueous or oiliness Suspension.These suspensions can be come according to techniques known in the art using appropriate dispersant or wetting agent and suspending agent Prepare.Aseptic injection preparation can also be aseptic injectable solution in nontoxic parenteral acceptable diluent or solvent or Suspension, such as solution in 1,3-BDO.The acceptable carrier and solvent that can be used is water, Ringer's solution and isotonic Sodium chloride solution.In addition, can typically use sterile fixed oil as solvent or suspension medium.

Therefore, any gentle fixed oil can be used, include the monoglyceride or diglyceride of synthesis.In injection Aliphatic acid, such as oleic acid and its glyceride ester derivatives can be used in the preparation of agent, can also use it is for example natural pharmaceutically Acceptable oils, such as olive oil or castor oil, particularly their polyoxyethylated versions.These oil solutions or suspension Can also include long-chain alcohol diluents or dispersant, for example carboxymethyl cellulose or pharmaceutically acceptable formulation include breast Agent and the similar dispersant commonly used in the preparation of suspension.Other conventional surfactants, such as Tweens, spans and Other emulsifying agents or bioavilability reinforcing agent commonly used in pharmaceutically acceptable solid, liquid or other formulations that prepare also may be used For preparing purpose.

The pharmaceutically acceptable composition of the present invention can be orally administered with any oral acceptable formulation, including But it is not limited to capsule, tablet, aqueous suspension or solution.In the case of oral tablet, conventional carrier include lactose and Cornstarch.Lubricant such as magnesium stearate can generally also be added.For the oral of capsule form, useful diluent includes Lactose and dried corn starch.When needing oral aqueous suspension, active component is mixed with emulsifying agent and suspending agent.If needed Will, some sweeteners, flavor enhancement or colouring agent can also be added.

Alternately, pharmaceutically acceptable composition of the invention can be used for rectally with suppository form.These Suppository can be prepared by the way that medicine is mixed with appropriate non-irritating excipient, and the excipient is solid at room temperature, But it is liquid under intestines temperature, therefore can in the rectum melts and discharge medicine.This material includes cocoa butter, beeswax and poly- second Glycols.

Liquid dosage forms for oral administration includes but is not limited to pharmaceutically acceptable emulsion, micro emulsion, solution, suspension, sugar Slurry and elixir.In addition to reactive compound, liquid dosage form can also contain inert diluent commonly used in the art, for example water or Other solvents；Solubilizer and emulsifying agent, such as ethanol, isopropanol, ethyl carbonate, ethyl acetate, phenmethylol, Ergol, Propane diols, 1,3-BDO, dimethylformamide, oils (is particularly cottonseed oil, peanut oil, corn oil, embryo oil, olive oil, castor Sesame oil and sesame oil), glycerine, tetrahydrofurfuryl alcohol, the fatty acid ester of polyethylene glycol and sorbitan, and their mixture. Besides inert diluents, the Orally administered composition can also include auxiliary agent, such as wetting agent, emulsifying agent and suspending agent, sweet taste Agent, flavouring and aromatic.

Injectable formulation (the aqueous or oil-based suspension of such as aseptic injection) can use what is be adapted to according to known technology Dispersant or wetting agent and suspending agent are prepared.Aseptic injection preparation can also be in nontoxic parenteral acceptable diluent Or the aseptic injectable solution in solvent, suspension or emulsion, such as the solution in 1,3-BDO.What can be used is acceptable Carrier and solvent have water, Ringer's solution, U.S.P. and isotonic sodium chlorrde solution.In addition, typically using sterile fixed oil It is used as solvent or suspension medium.To this end it is possible to use, any gentle fixed oil, includes the monoglyceride or glycerine of synthesis Diester.In addition, aliphatic acid, such as oleic acid can also be used in the preparation of injection.

Injectable formulation can be sterilized using preceding, for example, filtered by bacterial-retaining filter, or incorporation The bactericidal agent of sterile solid compositions form, the wherein sterile solid compositions can be sterile using being preceding dissolved or dispersed in In water or other sterile injectable mediums.

In order to extend the effect of the compounds of this invention, it is often necessary to delay suction of the compound after subcutaneously or intramuscularly injecting Receive.This can be realized using the crystal of poorly water-soluble or the liquid suspension of amorphous substance.The absorption rate of compound takes Certainly in its rate of dissolution, and the latter depends on crystal size and crystal formation.Alternately, compound is dissolved or is suspended in oil In class medium, the delay for realizing the compound form of parenteral absorbs.The depot formulation of injectable can by The microencapsule matrices of compound are formed in Biodegradable polymeric such as polylactide-co-glycolide to prepare.According to compound With the ratio and the property of used particular polymers of polymer, the rate of release of compound can be controlled.Other biologies can The example of degradation polymer includes poly- (ortho esters) and poly- (acid anhydrides).Depot injectable formulations can also be by by compound bag It is embedded in liposome or micro emulsion with tissue-compatible to prepare.The compound of the present invention can be mixed into other routine to delay Release formulation, to provide its controlled release within a few hours to a couple of days to the desired time limit of several months.

Compositions for rectal or vaginal administration is preferably suppository, they can by by the compounds of this invention with it is appropriate Nonirritant excipient or carrier such as cocoa butter, polyethylene glycol or suppository are prepared with wax mixing, the nonirritant figuration Agent or carrier are solid at ambient temperature, but are under body temperature liquid, therefore are melted in rectum or vaginal canal, are discharged Reactive compound.

Include capsule, tablet, pill, pulvis and granule for oral solid dosage forms., will in these solid dosage forms Reactive compound is mixed with least one inert pharmaceutically acceptable excipient or carrier, and the excipient or carrier are for example For sodium citrate or Dicalcium Phosphate, and/or a) filler or extender, such as starch, lactose, sucrose, glucose, mannitol And silicic acid, such as b) adhesive, carboxymethyl cellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and Arabic gum, c) Wetting agent, such as glycerine, such as d) disintegrant, agar, calcium carbonate, potato or tapioca, alginic acid, some silicate and Sodium carbonate, e) dissolves retarding agent, such as paraffin, f) adsorption enhancer, such as quaternary ammonium compound, such as g) wetting agent, cetanol And glyceryl monostearate, such as h) adsorbent, kaolin and bentonite, and i) lubricant, such as talcum powder, calcium stearate, Magnesium stearate, solid polyethylene glycol, NaLS and its mixture.For capsule, tablet and pill, formulation can also be wrapped Containing buffer.

Can also be using the solid composite of similar type as the filler in soft or hard filling capsule, and use tax Shape agent is such as lactose (lactose) or toffee (milk sugar) and macromolecule polyethylene glycol.Tablet, lozenge, capsule, ball The solid dosage forms such as agent and granule, which can be prepared into have, to be coated and shell, such as known in enteric coating and field of pharmaceutical preparations It is other to be coated.They can optionally containing opacifier or only or it is preferential in intestines and stomach certain part (optionally with The mode of delay) discharge active component composition.The example for the embedding composition that can be used includes polymeric material and wax class. Can also be using the solid composite of similar type as the filler in soft or hard filling capsule, and use excipient for example Lactose or toffee and high molecular weight polyethylene glycol etc..

Reactive compound can also be the form of microencapsulation, and contain one or more excipient as described above.Piece The solid dosage forms such as agent, lozenge, capsule, pill and granule can be prepared into coating and shell, such as enteric coating, release control Property processed is coated to be coated with other known to field of pharmaceutical preparations.In these solid dosage forms, can by reactive compound with least A kind of inert diluent, such as sucrose, lactose or starch mixing.Under normal circumstances, these formulations can also include and remove inertia Other materials beyond diluent, such as tableting lubricant and other compression aids, such as magnesium stearate and microcrystalline cellulose.It is right In capsule, tablet and pill, these formulations can also include buffer.They can optionally containing opacifier or Only or preferential certain a part of (optionally in a delayed fashion) discharge active component in intestines and stomach composition.It can use The example of embedding composition includes polymeric material and wax class.

The part or transdermal administration of the compound of the present invention include ointment, and paste, emulsion (creme), lotion coagulates Jelly, pulvis, solution, spray, inhalant or patch.As needed, aseptically by active component with pharmaceutically may be used The carrier of receiving and the mixing of any desired preservative or buffer.Ophthalmic preparation, auristilla and eye drops are also covered by this hair In bright scope.In addition, the present invention covers the use of transdermal patch, they have control compound to body deliver it is additional excellent Point.This kind of formulation can be prepared by the way that compound is dissolved or dispersed in appropriate medium.Influx and translocation can also be used Agent with increase compound pass through skin flux.Can be by providing rate controlling membranes or compound being dispersed in into polymer matrix Carry out speed control in matter or gel.

The compound of the present invention is preferably formulated to unit dosage forms, in favor of being administered and keeping the uniformity of dosage.This paper institutes Phrase " unit dosage forms " refers to the physical separation unit for being adapted to the medicine for the patient to be treated.It is understood, however, that this The compound of invention and every total daily dose of composition are determined by attending doctor in rational medical judgment scope.It is any The specific effective dose level of specific patient or organism depends on several factors, including the disease to be treated and disease Seriousness；The activity of used particular compound；Used concrete composition；The age of patient, body weight, general health shape Condition, sex and diet；The discharge rate of administration time, method of administration and used particular compound；The duration for the treatment of； Combine with used particular compound or medicine used at the same time, and similar factor known to medical domain.

It can mix different come the amount for the compounds of this invention for producing the composition in single formulation with carrier mass, Depending on the host treated, specific administering mode.It is preferred that, should compositions formulated allow to 0.01-100mg/kg The inhibitor of body weight/day, such as 0.1-100mg/kg and 0.1-10mg/kg body weight/day dosage, which is given, receives these compositions Patient.

According to the specific illness or disease to be treated or prevented, can also in the present compositions exist treatment or Prevent the other therapeutic agent that would generally be used during the illness.As used herein, treat or prevent a kind of disease specific or The other therapeutic agent that would generally be applied during illness is considered as " being adapted to the disease or illness to be treated ".Hereinafter provide The example of other therapeutic agent.

The content of other therapeutic agent in the compositions of the present invention no more than generally it comprising the therapeutic agent as unique Content in the composition of activating agent.Preferably, the content of therapeutic agent other present in composition disclosed by the invention It is the about 50%-100% of its usual content in the composition comprising the therapeutic agent as sole therapy activating agent.

The purposes of the compounds of this invention and composition

In one aspect of the invention, the invention is characterised in that the illness or disease for the treatment of PI3K γ mediations or mitigation institute State illness or the method for disease severity.Term " disease of PI3K γ mediations " used herein refers to that wherein known PI3K γ are same Any disease and other adverse conditions that drum works.

Therefore, in further embodiment, the compounds of this invention selective depression PI3K γ-isoform.At one In embodiment, the compounds of this invention or composition test in vitro in at least 20 times selectively relative to the same works of PI3K α Type selective depression PI3K γ-isoform.In another embodiment, PI3K gamma selectives compound of the invention is in vitro With at least 20 times selectively relative to each α in experiment, β and δ isoforms suppress γ isoforms.Present invention additionally comprises use this Invention compound and the selectivity of composition treat the patient that this kind for the treatment of needs in vivo.

The compound or composition of the present invention can be given together with one or more other therapeutic agents, wherein other Therapeutic agent is applied to disease to be treated, and other therapeutic agent and the compound or composition of the present invention are together as single Formulation is given, or the part as dosage is separately given with the compounds of this invention or composition.Other therapeutic agent can With from the present invention compound simultaneously or asynchronously give.In latter instance, it can be that 12 is small according to such as 6 hours to give When, 1 day, 2 days, 3 days, 1 week, 2 weeks, 3 weeks, progress of staggering for 1 month or 2 months.

The invention provides a kind of method for suppressing PI3K γ kinase activities in biological sample, this method is included biological sample Product are contacted with the compound or composition of the present invention.Term " biological sample " used herein refers to the sample outside live organism, Including but not limited to cell culture or its extract；Derived from the biopsy material of mammal or its extract；And blood, saliva Liquid, urine, excrement, seminal fluid, tear or other body fluid or its extract.Kinase activity in biological sample, particularly PI3K γ The suppression of kinase activity can be used for a variety of purposes well known by persons skilled in the art.The example of these purposes includes but is not limited to life Thing sample is preserved and bioassay.In one embodiment, the method for suppressing PI3K γ kinase activities in biological sample is limited In non-treatment method.

The preparation of the compounds of this invention

All abbreviations used herein, symbol and defined implication are consistent with those used in the scientific literature in the present age. See, e.g. Janet S.Dodd, ed., ACS Style Guide：A Manual Authors and Editors, second edition, Washington, D.C.：American Chemical Society, 1997.It is defined below describe term used herein and Abbreviation：

ATP adenosine triphosphates

The Brine saturation NaCl aqueous solution

CRED carbonyl reductases

DCM dichloromethane

DIEA diisopropylethylamine

DMA dimethyl acetamides

DMAP DMAPs

DMF dimethylformamides

DMSO dimethyl sulfoxides

dppfPdCl₂The chloro- palladium of 1,1 '-bis- (diphenylphosphino)-ferrocene two

DTT dithiothreitol (DTT)s

ESMS electron spray mass spectrometries

Et₂O ether

EtOAc ethyl acetate

EtOH ethanol

HEPES 4- (2- hydroxyethyls) -1- piperazine ethanesulfonic acids

HPLC high performance liquid chromatographies

LC-MS C/MS (liquid chromatography-mass spectrography)s

The m- chlorine benzylhydroperoxide of mCPBA

Me methyl

MeOH methanol

MTBE methyl tertiary butyl ether(MTBE)s

MC methylcellulose

NAD NADHs

NMP 1-METHYLPYRROLIDONEs

Ph phenyl

RTorrt room temperatures (20 DEG C -25 DEG C)

The tBu tert-butyl groups

TBME t-butyl methyl ethers

TCA trichloroacetic acids

THF tetrahydrofurans

TEA triethylamines

Synthetic method

Typically can be by method described herein or by preparing chemical combination well known to a person skilled in the art other methods Thing 1.

The chloro- 6- of embodiment 1.2- (1- (fluoro ethyls of 2,2- bis-) -1H- pyrazoles -4- bases) -4,7,7- trimethyl -6,7- dihydros - The preparation of 5H- pyrrolo-es [3,4-b] pyridine -5- ketone (compound 1006)

Scheme 1

As shown in the step 1-i of scheme 1, to 2,4- lutidines -3- Ethyl formate (chemical combination in nitrogen atmosphere Thing 1001,20.2g, 112.5mmol) the chloro- 1,3,5- tri- of 1,3,5- tri- are gradually added in solution in dichloromethane (100mL) Piperazine (triazinane) -2,4,6- triketones (31.4g, 135.0mmol) last 15 minutes.The reactant mixture is stirred in room temperature Mix 18 hours.The white precipitate being filtrated to get, then uses saturation NaHCO successively₃The aqueous solution (2x100mL) and salt solution (100mL) Wash filtrate.Dry organic phase (Na₂SO₄), filter, be concentrated in vacuo, obtain 2- (chloromethyl) -4- methylnicotinic acids ethyl ester (22.9g, Compound 1002), it is yellow oil：ESMS (M+H)=213.96.Then the material is used for without further purification next Step：

As shown in the step 1-ii of scheme 1, to 2- (chloromethyl) -4- methylnicotinic acids ethyl ester (compound 1002, 112.0g, 524.2mmol) add in solution in dichloromethane (484mL) 3- chloro peroxide acids (141.0g, 629.0mmol).The reactant mixture is stirred overnight in nitrogen atmosphere in room temperature.This is diluted with dichloromethane (200mL) to mix Compound, uses saturation NaHCO successively₃The aqueous solution (100mL), saturation Na₂CO₃The aqueous solution (100mL 2M solution) and salt water washing. Dry organic phase (Na₂SO₄), filter, be concentrated in vacuo, obtain 2- (chloromethyl) -3- (carbethoxyl group) -4- picolines -1- oxidations Thing (compound 1003)：ESMS (M+H)=230.25.Then the material is used for next step without further purification：

As shown in the step 1-iii of scheme 1,2- (chloromethyl) -3- (carbethoxyl group) -4- picolines 1- is aoxidized The solution of thing (compound 1003,69.3g, 301.8mmol) in phosphoryl chloride phosphorus oxychloride (450.1mL, 4.8mol) is stirred in nitrogen atmosphere Mix, heated 60 hours at 95 DEG C.The reactant mixture is cooled to room temperature, phosphoryl chloride phosphorus oxychloride is evaporated in vacuo out.It is dark residual by what is obtained Excess is dissolved in dichloromethane (100mL), is poured on the ice in 1L beakers (500g).Obtained mixture is stirred 10 minutes. Use saturation NaHCO₃The aqueous solution adjusts the pH of the mixture to a little higher than 7.Organic phase is separated, then uses dichloromethane extraction water Phase.The continuous organic phase merged with salt water washing, uses Na₂SO₄Dry, filter, be concentrated in vacuo.Use EtOAc/ hexanes (1:3) make Residue is removed under reduced pressure and 80% pure product is obtained after volatile materials (as basis by silicagel pad¹Shown in HNMR analyses ).Material (the 10-25%EtOAc/ hexanes, 330g Teledyne ISCO is further purified by silica gel medium pressure chromatography Post), obtain 6- chloro- 2- (chloromethyl) -4- methylnicotinic acids ethyl esters (compound 1004,36.5g):ESMS (M+H)=248.04.

As shown in the step 1-iv of scheme 1, to 1- (2,2- bis- fluoro ethyl) pyrazoles -4- amine (48.2g, 6- chloro- 2- (chloromethyl) -4- methylnicotinic acid ethyl ester (compounds 327.5mmol) are added in the solution in isopropanol (1.7L) 1004,65.0g, 262.0mmol), then add DIPEA (45.6mL, 262.0mmol).The reaction is mixed Thing is heated 72 hours at 55 DEG C.Obtained white thick suspension is cooled to room temperature, filtered, then with isopropanol (200mL) and Ether (500mL) is washed.Obtained solid is dried overnight in 50 DEG C of vacuum drying ovens, the chloro- 6- of 56g2- (1- (2,2- difluoros are obtained Ethyl) -1H- pyrazoles -4- bases) -4- methyl -6,7- dihydro -5H- pyrrolo-es [3,4-b] pyridine -5- ketone (compound 1005):¹HNMR (300MHz, DMSO-d6) δ 8.28 (s, 1H), 7.87 (d, J=0.5Hz, 1H), 7.52 (s, 1H), 6.37 (tt, J= 54.9,3.7Hz, 1H), 4.85 (s, 2H), 4.67 (td, J=15.2,3.7Hz, 2H), 2.65 (s, 3H).The material includes 7% Non-cyclizing accessory substance [the chloro- 2- of 6- (((1- (fluoro ethyls of 2,2- bis-) -1H- pyrazoles -4- bases) amino) methyl) -4- methylnicotinic acids Ethyl ester], it is used as such for subsequent reaction.

As shown in the step 1-v of scheme 1, to the chloro- 6- of 2- (1- (2,2- bis- fluoro ethyl) -1H- pyrazoles -4- bases) -4- Methyl -6,7- dihydro -5H- pyrrolo-es [3,4-b] pyridine -5- ketone (compound 1005,46.0g, 147.1mmol) is in DMF Iodomethane (20.1mL, 323.6mmol) is added in solution in (782.0mL).The mixture is cooled to 5 DEG C, is gradually added Sodium hydride (12.9g, 323.6mmol 60% dispersion oil), lasts 15 minutes.By the reactant mixture in 3 DEG C of stirrings 45 minutes.HPLC analyses show that monomethyl (10%), two-methylate (74%) and three-methylate (15%) product with consuming Raw material mixture.Again after 1 hour, sodium hydride (1.29g, 32.36mmol 60% dispersion oil) is added, HPLC analyses display 84% 2-methylate desired product and 16% 3-methylated by-product.By adding saturation NH₄Cl water Solution (1L), sodium thiosulfate (400mL) and water (1L) terminating reaction.Aqueous phase is extracted 2 times with EtOAc (800mL).Dry and close And organic phase (Na₂SO₄), filter, be concentrated in vacuo.Residue (0-40%EtOAc/ hexanes are purified by silica gel medium pressure chromatography Gradient, uses 800g Teledyne ISCO posts), the chloro- 6- of 2- (1- (2,2- bis- fluoro ethyl) -1H- pyrazoles -4- bases) -4 are obtained, 7,7- trimethyl -6,7- dihydro -5H- pyrrolo-es [3,4-b] pyridine -5- ketone (compound 1006,24g), are white solid：¹HNMR(400MHz,DMSO-d6)δ8.21(s,1H),7.83(s,1H),7.54(s,1H)6.58-6.25(m,1H),4.67 (td, J=15.1,3.7Hz, 2H), 2.65 (s, 3H), 1.51 (s, 6H)

Embodiment 2：(R) -6- (1- (fluoro ethyls of 2,2- bis-) -1H- pyrazoles -4- bases) -4,7,7- trimethyls -2- (5- (2,2, The fluoro- 1- hydroxyethyls of 2- tri-) pyridin-3-yl) -6,7- dihydro -5H- pyrrolo-es [3,4-b] pyridine -5- ketone (compound 1) system It is standby

Scheme 2

As shown in the step 2-i of scheme 2, water (10L) is added in 20L reactors, KH is then added₂PO₄ (136g).The mixture is stirred uniform to reaching, 0.1M KH are obtained₂PO₄Buffer solution.This is buffered by adding 2M NaOH The pH of liquid is adjusted to pH7.5.Internal temperature is set to reach 37 DEG C.Taking out about 500mL buffer solutions is used to then add NAD and CRED A131 cytoplasms (cellpaste) (Almac Group, Ltd.).Therefore, it is NAD (20g) is molten in buffer solution (100mL) Liquid is added in remaining 9.5L buffer solutions, then adds 1- (5- bromopyridine -3- bases) -2,2,2- trifluoro second -1- ketone (1020g) Solution in MTBE (1.5L).Isopropanol (1L) is used to rinse flask again into reaction.By by CRED Suspensions of the A131cellpaste (100g) in buffer solution (400mL) is added in the reactant mixture of the stirring and started also It is former.In the course of reaction, biphasic reaction sample (~2mL) is taken out from the mixture of stirring, is extracted with EtOAc (10mL), Use MgSO₄Dry, evaporation passes through¹HNMR is analyzed in DMSO-d6, to monitor conversion ratio of the raw material to product.At 1 hour Afterwards, it was observed that 50% conversion ratio.After 3 hours, it was observed that>99% conversion ratio.With 2M NaOH by the reaction system PH is adjusted to 11, is stirred 30 minutes, so that enzyme denaturation.PH is adjusted to 9 with 2M HCl.Added into the reactant mixture EtOAc (5L) and diatomite (650g), are persistently stirred 10 minutes.The emulsion being filtrated to get by Celite pad, has to separate Machine phase and aqueous phase.With EtOAc (2L) Rubbing pad for washing use, organic layer is separated.With EtOAc (6L) aqueous layer extracted, conjunction is washed with salt solution (4L) And organic phase, by adding MgSO₄(200g) and stirring drying in 30 minutes.Filtering, is removed in vacuum volatile materials, obtains light Yellow oil, the rapid curing when standing.Solid is smashed blocking, slurry is stirred into hexamethylene (2L).To the mixing EtOAc (~500mL) is added in thing, stirs to dissolve the solid at 45 DEG C.Then the mixture is concentrated in vacuo again, until white Solid starts to precipitate from solution.Hexamethylene (2L) is added, the solution is cooled to 0 DEG C with ice bath.After stirring 30 minutes, lead to Filtering collection solid, is washed, vacuum drying oven is dried, and obtains 812g (R) -1 with hexamethylene (500mL)) -1- (5- bromopyridines -3- Base) -2,2,2- trifluoro second -1- alcohol (compound 1007,99.95%ee, analyzed by HPLC) are graininess white solid：¹HNMR(400MHz,DMSO-d6)δ8.72(s,1H),8.61(s,1H),8.08(s,1H),5.09-5.15(m,1H).

As shown in the step 2-ii of scheme 2, to (1R) -1- (the bromo- 3- pyridine radicals of 5-) -2,2,2- trifluoro-ethanols (are changed Compound 1007,49.5g, 193.3mmol), the 4,4,5,5- tetramethyls -2- (boron of 4,4,5,5- tetramethyl -1,3,2- dioxanes penta Alkane -2- bases) -1,3,2- dioxaborolanes (58.9g, 232.0mmol) and KOAc (37.9g, 386.6mmol) dioxanes Solution in (1.2L) leads to nitrogen 20 minutes.DppfPdCl is added into the reactant mixture₂*DCM(7.8g,9.7mmol).Again Lead to nitrogen 20 minutes to the mixture, be heated to backflow 2 hours.It is cooled to after room temperature, passes through florisil pad (400mL) mistake The mixture is filtered, 50%EtOAc/CH is used₂Cl₂(1.5L) washs pie.The filtrate being concentrated under reduced pressure to give, obtains yellow oily Thing, is diluted with hexane (800mL), is concentrated in vacuo, is obtained foam yellow solid.By the yellow solid and hexane (800mL) one Stirring 2 hours is played, white solid precipitation is obtained.By filtering collection white solid, dry, obtain (1R) -2,2,2- tri- fluoro- 1- [5- (4,4,5,5- tetramethyl -1,3,2- dioxaborolanes -2- bases) -3- pyridine radicals] ethanol (compound 1008, 48.4g)：¹HNMR(400MHz,CDCl₃)δ8.97(s,1H),8.78(s,1H),8.29(s,1H),5.10-5.21(m,1H)； 1.38(s,6H),1.29(s,6H).

As shown in the step 2-iii of scheme 2, to the chloro- 6- of 2- (1- (2,2- bis- fluoro ethyl) -1H- pyrazoles -4- bases) - 4,7,7- trimethyl -6,7- dihydro -5H- pyrrolo-es [3,4-b] pyridine -5- ketone (compound 1006,42.0g, 123.3mmol), The fluoro- 1- of (1R) -2,2,2- three [5- (4,4,5,5- tetramethyl -1,3,2- dioxaborolanes -2- bases) -3- pyridine radicals] ethanol (compound 1008,46.7g, 148.0mmol) and Na₂CO₃(28.8g, 271.3mmol) is in DMF (630mL) and water (210mL) Solution in lead to nitrogen 30 minutes.DppfPdCl is added into the mixture₂* DCM (2.99g, 3.699mmol), gives the mixing Thing leads to nitrogen 30 minutes again.The reactant mixture is heated to 103 DEG C, stirred 2 hours.The mixture is cooled to room temperature, used Water (2L) dilutes.Aqueous phase is extracted 2 times with EtOAc (1L).The organic phase of merging is concentrated in vacuo under high vacuum, to remove DMF. Residue is diluted with EtOAc, is washed with water, then uses salt water washing.Dry organic phase (Na₂SO₄), filter, be concentrated in vacuo.It is logical Crossing silica gel medium pressure chromatography purifying thick residue, (0-100%EtOAc/ hexane gradients use 1500g Teledyne ISCO Post), the desired products of 54g are obtained, are pale red foaming solid.The solid is dissolved in dichloromethane.Press through florisil Pad (200mL), EtOAc/CH is used successively₂Cl₂Mixture [first 40% (1L), then 60% (1L), then 80% (1L)] is washed Wash.Merging filtrate, is concentrated in vacuo.Residue is diluted 2 times with heptane (400mL), is concentrated in vacuo, obtains pie, Ran Houyong TBME is washed, faint yellow to remove, and is dried 4 days in 60 DEG C of vacuum drying ovens, is obtained (R) -6- (1- (2,2- bis- fluoro ethyl) -1H- pyrroles Azoles -4- bases) -4,7,7- trimethyl -2- (5- (2,2,2- tri- fluoro- 1- hydroxyethyls) pyridin-3-yl) -6,7- dihydro -5H- pyrroles And [3,4-b] pyridine -5- ketone (compound Isosorbide-5-Nitrae 7g)：¹HNMR (400MHz, CDCl₃) δ 9.33 (s, 1H), 8.75 (s, 1H), 8.54 (s, 1H), 8.01 (s, 1H), 7.75 (s, 1H), 7.62 (s, 1H), 6.14 (tt, J=55.4,3.6Hz, 1H), 5.28-5.11 (m, 1H), 4.51 (td, J=13.5,4.2Hz, 2H), 4.33 (d, J=4.6Hz, 1H), 2.82 (s, 3H), 1.68 (s, 6H)

To the external efficiency of PI3K- γ lipid kinases

Embodiment 3.PI3K suppresses experiment

Using the Biomek FX from Beckman Coulter, by the sheet of 2.5 times of serial dilutions in 10 100%DMSO Each 1.5 μ L of invention compound are added to 96 hole polystyrene flat boards [Corning, Costar Item No.3697] single hole In (hereinafter referred to " instrument connection ").One instrument connection is also free of the DMSO of compound containing 1.5 μ L.Another hole contains in DMSO There is the inhibitor for the concentration for being known to completely inhibit enzyme (hereinafter referred to " background hole ")., will using Titertek Multidrop 50 μ L reactant mixtures [100mM HEPES pH 7.5,50mM NaCl, 10mM DTT, 0.2mg/mL BSA, 60 μM of phosphatidyls Inositol (4,5) bisphosphate diCl6 (PI (4,5) P₂；Avanti Polar Lipids, Cat.No.840046P) and concern PI3K isoforms (concentration of isoform is referring to table 1)] it is added in each hole.To start reaction, by 50 μ L ATP mixtures [20mM MgCl₂, 6 μM of ATP (100 μ Ci/ μm ol³³P-ATP)] be added in each hole, then by each hole at 25 DEG C it is warm Educate 30 minutes.The ultimate density in each hole is 50mM HEPES 7.5,10mM MgCl₂, 25mM NaCl, 5mM DTT, 0.1mg/ ML BSA, 30 μM of PI (4,5) P₂, the PI3K isoforms of 3 μM of ATP and concern (referring to table 3).Final compound is dense in each hole Spend for 10 μM of -1nM.

Table 1

PI3K isoform concentration	PI3K-α	PI3K-β	PI3K-γ	PI3K-δ
					Enzyme concentration in reactant mixture	4nM	20nM	4nM	4nM
Final enzyme concentration	2nM	10nM	2nM	2nM

After incubation, the reaction in each hole is quenched by adding 50 μ L stop baths [30%TCA/ water, 10mM ATP]. Then the reactant mixture that each is quenched is transferred to [Corning, Costar Item in 96 hole glass fibre filter plates No.3511].Planar vacuum is filtered, then with 150 μ L 5%TCA/ water improvement Bio-Tek Instruments Washed 3 times in ELX-405 automatic flat-plate washers.50 μ L scintillation fluids are added in each hole, and by flat board in Perkin- Elmer TopCount^TMReading on NXT liquid scintillation counters, inhibiting value is represented so as to obtain³³P- numbers.

The numerical value in background hole is subtracted in the numerical value obtained from each instrument connection, data are fitted into Morrison and Stone, Comments Mol.Cell Biophys.2：347-368, the competition described in 1985 is combined closely in Ki equatioies.Compound 1 PI3K γ inhibition levels linearly change with ATP concentration, show Reverse transcriptase, wherein Ki values be 8+/- 4nM.In addition, also It was observed that compound 1 is to PI3K- α, PI3K- β, the Isoform selective of PI3K- δ isoforms test and determines to 15 times of PI3K γ Selectivity above, as shown in table 2.

The PI3K Isoform selectives of the compound 1 of table 2.

Cell efficiency

The multi-subunit complex that PI3K is made up of regulation subunit and catalytic subunit.This quasi-enzyme catalytic phosphatidylinositols -4, The phosphorylation of 5- diphosphonic acid (PIP2), obtains second messenger's phosphatidylinositols -3,4,5- triphosphoric acids (PIP3).Receptor activation causes PIP3 levels are instantly increased.PIP3 works as the docking site on plasma membrane, so as to supplement and activate comprising plek homotropic The protein (such as Akt, PDK-1, Tek kinases) of albumen homology (PH) domain.Then they adjust key cells work( Can, such as growth, metabolism, migration, respiratory burst.Due to downstream effect thing in PI3K signal transductions be in it is shared, What which kind of PI3K isoform decision raised in activation is acceptor.Based on this, it is numerous based on biochemistry-pAkt's and functional trial For analyzing efficiency and selectivity of the PI3K gamma inhibitors relative to other PI3K isoforms.

The pAkt that MCP-1 is stimulated in embodiment 4.THP-1 cells

Chemotactic factor (CF) such as MCP-1 combines its acceptor, causes PI3K γ signal transductions by way of activation.PI3K γ cause PIP3 to give birth to Activated into downstream molecules (such as PDK-1 and Akt).Akt phosphorylation is that PI3K activity is measured in cell.In this experiment, make THP-1 cells (human monocyte cell line) overnight starvation, to exhaust pAkt levels.Then being stimulated with MCP-1 causes PI3K γ in 3 minutes Akt phosphorylation on the threonine 308 of induction and the site of serine 473.Fixed cell, dyeing intracellular phosphoAKT (Ser473), then using BD FACSCalibur^TMCell counter is analyzed, and obtains the measured value of cell PI3K gamma activities. Compound 1 has 0.24 ± 0.07 μM of IC in this experiment₅₀.Referring to table 3.

Efficiency of the compound 1 of table 3. in PI3K- α/βs/δ dependence in vitro tests

The whole blood of embodiment 5. and leucocyte oxidative burst Experiment on Function

Neutrocyte, monocyte and macrophage are the key mediators of natural immunity.They discharge active oxygen (ROS) as the part of congenital immunity inflammatory response.ROS is produced and induced by mediator of inflammation, such as chemotactic factor (CF), bacterial peptide Class and the complement of recruitment congenital immunity cell to inflammation part.These mediator of inflammation triggering PI3K γ are activated and are started downstream Signal transduction cascade, it causes the complete nadph oxidase compound assembling at generation ROS plasma membrane.It is true according to the ROS of generation Determine the PI3K γ functional activities in whole blood and buffycoat neutrocyte and monocyte.With TNF-α by cytositimulation 10 minutes, Then stimulated 20 minutes with chemotactic peptide fMLP derived from bacteria cell wall, and load non-fluorescence dyestuff dihydro rhodamine 1,2, 3(DHR).Thus DHR is oxidized to fluorescence rhodamine by the ROS produced by cell, causes the fluorescence rhodamine content in cell to increase Plus.According to neutrocyte and monocyte, generation ROS ability determines PI3K γ functional activities after fMLP stimulations.Use BD FACSCalibur^TMLeucocyte and whole blood sample are analyzed, so as to the cell quantification being positive to fluorescence rhodamine.Pale brown confluent monolayer cells IC is generated in the presence of serum_50S, and whole blood test generates IC under in the absence of serum_50S.Compound 1 is thin in vain in buffycoat There is 0.22 μM of IC in born of the same parents' experiment₅₀And there is 0.57 μM of IC in whole blood test₅₀, referring to table 3.

The pAkt that CSF-1 is stimulated in embodiment 6.THP-1 cells

Growth factor, cell factor and other receptor tyrosine kinase parts (such as CSF1) combine its acceptor, cause IA classes PI3K signal transductions are by way of activation.PI3K activation causes generation PIP3 and downstream molecules (such as PDK-1 and Akt) activation.Akt phosphoric acid Change is that PI3K activity is measured in cell.THP-1 cells (human monocyte cell line) overnight starvation is set to exhaust pAkt levels.With CSF-1 stimulates the phosphorylation of Akt on the threonine 308 for causing PI3K α/βs/δ inductions for 5 minutes and the site of serine 473.It is fixed thin Born of the same parents, dyeing intracellular phosphoAkt (Ser473), then using BD FACSCalibur^TMCell counter is analyzed.This is cell The measured value that IA classes PI3K suppresses.Compound 1 has in this experiment>9.7 μM of IC₅₀, it is shown in IA classes PI3Ks selection Property.Referring to table 4.

The human B cell proliferation test of embodiment 7.

The development of B cell and active height depend on PI3K δ.PI3K γ are by B-cell receptor (BCR) compound in signal There is required and non-unnecessary effect in conductive process.The Ca+ of IgM inductions, which is flowed into and bred, to be lacked PI3K- δ or is using Weaken in the mouse of PI3K- δ inhibitor.PI3K γ do not work in any B cell activity.BCR compounds are to PI3K γ The specificity of isoform causes it to turn into the preferable PI3K γ experiments for being used to evaluate PI3K γ inhibitory action degree in cell. The human B cell of purifying is stimulated with anti-IgM in the presence of test compound.After 4 days, measure thin using cell titer-glo Born of the same parents' survival rate/proliferation rate, to determine the ATP contents of cell in each hole.It is PI3K γ inhibitory action journeys that propagation, which lacks or reduced, The measured value of degree.Compound 1 has 3.05 ± 0.44 μM of IC₅₀, show 11- times of PI3K- δ selective window.Referring to table 4.

Embodiment 8.HUVEC proliferation tests

PI3Ks is that the signal of interest in the numerous growth factor downstreams for adjusting cell survival, growth and cell cycle entrance is passed Lead molecule.The PI3K- α and PI3K- β isoforms regulation cell cycle enters；Suppressing any isoform causes cell growth to decline. HUVECs is the primary human umbilical vein's endothelial cell for expressing PI3K α and PI3K β isoforms.Suppress PI3K α or PI3K β isoforms to appoint It is a kind of or both will suppress HUVECs cell growths.Compound is sprawled on HUVECs, 96 hours after compound processing, The ATP contents of cell in each hole are determined using cell titer-glo, to measure cell proliferation rate/survival rate.Propagation lacks Or it is measuring for PI3K α and/or PI3K β inhibitory action degree to reduce.Compound 1 has in this experiment>19 μM of IC₅₀, show Show>The selectivity of 74- times of PI3K α/β.Referring to table 4.

Embodiment 9.MCF-7 proliferation tests

PI3K-Akt signal transductions are bred, survive and grown by way of many Normal cellular processes of regulation, including cell, they It is crucial for tumour generation.PI3K signal transductions generally occur in people's cancer by way of imbalance.MCF7 is human breast carcinoma Cell line, it has activation E545K PI3K α heterozygous mutants in the helix domain of p110 α albumen, causes PI3K- α approach Hyperactivity hyperkinesia.The PI3K- alpha signals conduction suppressed in MCF7 cells suppresses cell growth and it is possible thereby in MCF7 proliferation tests Evaluate PI3K- alpha inhibitors.Compound is added in MCF7 cells, 96 hours after compound processing, cell titer- are used Glo determines the ATP contents of cell in each hole, to measure cell proliferation rate/survival rate.It is PI3K α suppressions that propagation, which lacks or reduced, Effect degree processed is measured.Compound 1 has in this experiment>17 μM of IC₅₀, thus show>67- times of PI3K α selection Property.Referring to table 4.

As described above, compound 1 has splendid efficiency, it is 0.25 μM in the experiment of PI3K γ relevant cells；Based on There is 12- times of PI3K- δ selectivity in the experiment of cell；With 40-80 times of PI3K α and PI3K β selectivity.

Efficiency of the compound 1 of table 4. in PI3K α/βs/δ dependence in vitro tests

* each test data (μM)->20.>20,19.8

The each test datas (μM) -15.7 of *,>20,18,17

Drug metabolism

Compound 1 is metabolized caused by the recombinant C YP enzymes of embodiment 10.

Microsome comprising recombinant C YP enzymes (CYP1A2,2B6,2C8,2C9,2C19,2D6,2E1 and 3A4) is used for which to be determined Plant the oxidation of CYP catalytic cpds 1.Therefore, by compound 1 (1 μM) and each recombinant C YPs one in the presence of co-factor NADPH Rise and incubate.Remaining parent drug percentage is determined at the end of incubation period by LC-MS/MS and the percentage that exists during with starting Frequently compared with.As a result it is as shown in table 5.Despite with low apparent rate, but chemical combination is only detected in being incubated together with CYP3A4 The metabolism of thing 1.

The stability * that the compound 1 of table 5. is incubated together with recombinant C YP enzymes

* data are expressed as average value (SD)

Suppression of the compound 1 of embodiment 11. to CYP enzymes

In appraiser's hepatomicrosome compound 1 (0.01-100 μM) reversibly suppress CYP enzymes (CYP1A2,2B6,2C8, 2C9,2C19,2D6 and 3A4) potential.Suppress to test to use with specific substrates concentration (close to its dissociation constant (Km) value) and be directed to The selective CYP probes of every kind of CYP enzymes are carried out.Data are as shown in table 6.Compound 1 is CYP1A2,2B6 and 2C8 moderate suppression Preparation；IC₅₀It is worth for 4-7 μM.

The suppression of table 6. compound, 1 pair of CYP enzyme in people's hepatomicrosome

IC is used also in people's hepatomicrosome₅₀Transfer Experiment evaluation is pressed down using compound 1 to CYP3A4 time dependence System.In our current research, by 0.1-50 μM of compound 1 together with people's hepatomicrosome is in the presence of NADPH and in the absence of it Precincubation 0 and 30 minutes.Then incubation system is diluted 10- times with buffer solution and determines remaining CYP3A4 (testosterone -6- β-hydroxyl Change enzyme) activity, last 10 minute time limit.The IC of compound 1 in the presence of NADPH or in the absence of it₅₀There is no conspicuousness to change Become (IC₅₀Change=1).

This result is verified in follow, wherein being evaluated after the precincubation together with 10 or 50 μM of compounds 1 M- dependence suppresses during CYP3A4 in people's hepatomicrosome, last the time limit for 0,5,10,15 and 30 minutes.In this research, two Under kind of concentration, compared with the Kobs=0.053/min of positive control mifepristone, it was observed that CYP3A4 activity missings speed Constant (Kobs) is 0.0039/min.The data display compound 1 of two results from these researchs is not with time dependence Mode suppresses CYP3A4.

The enzyme induction of embodiment 12.

In the DPX2 cells for Bel7402 the activation pregnane of compound 1-X is evaluated with 0.1-30 μM of 6 concentration People PXR genes and the luciferase for two promoters for being connected to people's CYP3A4 genes in the potential of acceptor (PXR), the cell line Reporter is stably overexpressed.Rifampin is used as positive control, compound 1 shows the not notable activated water for obtaining response Flat, it is with EC50 values>The 5% of 30 μM of positive control.

The potential that the compound 1 in liver cell induces CYP1A2 and CYP3A4 is also evaluated.By 0.1-30 μM of compound 1 48 hours, and and positive control are incubated together with the stored refrigerated primary human hepatocyte from 3 different donors of culture Effect compare (i.e. CYP inducers：50 μM of Omeprazoles, CYP1A2；With 10 μM of rifampins, CYP3A4).By using LC- The metabolin formation of MS/MS monitoring specific C YP probe substrates determines CYP active (phenacetin, CYP1A2；And testosterone, CYP3A4).Analyze CYP messenger RNA (mRNA) to confirm CYP inductive potencies by RT-PCR.As a result it is as shown in table 7.

After compound 1 in all CYP1A2 or CYP3A4 activity and mRNA level in-site in 3 liver cell lot numbers Change is below the 20% of positive control.These vitro datas show that behind in human liver cell 48 hours compound 1 has Some induction CYP1A2 or CYP3A4 potential is relatively low.

CYP1A2 and CYP3A4 induction of the compound 1 of table 7. in human liver cell

^aIt is expressed as the multiple change relative to solvent control

NR¹Do not report, because response is less than excipient control

NR²Do not report, because cytotoxicity

The potential permeability of the effluent of embodiment 13.

The permeability of compound 1 is evaluated using Caco-2 and MadinDarby dogs kidney (MDCK) wild-type cell system.Make thin Born of the same parents' (permeability on measurement A- to-B directions) or bottom outside (permeability on measurement B- to-A directions) on top surface Medicine in buffer solution and incubated 1 hour at 37 DEG C.As a result it is as shown in table 7.In A- in MDCK and Caco-2 cell lines Higher (the respectively 33 and 18x10 of permeability on to-B directions^-6Cm/ seconds).

Whether it is effluent transport protein bottom to compound 1 by user P-gp (MDR) the mdck cell systems overexpressed Thing is evaluated.Excipient transhipment (discharge rate=35.1) is detected in the cell line, it is P-gp substrates to show compound 1.Ginseng It is shown in Table 8.

The general introduction that discharge rate and compound 1 are reclaimed in table 8.Caco-2, MDCK-WT and MDCK-MDR1 cell line

Internal pharmacokinetics

The intravenous push of embodiment 14. is administered

It is intravenous apply single bolus dose after, compound 1 in all test approaches have low systemic clearance and Long half-lift.Referring to table 9.Clearance rate value of the compound 1 in the hepatic blood flow of mouse, rat, dog and monkey is represented 5.4%th, 3.6%, 13% and 26%.Volume of distribution is more than whole body water volume, shows that compound 1 is distributed in tissue.

The single IV of table 9. injects the mean serum pharmacokinetic parameter using rear compound 1

^aHBF, hepatic blood flow

^bThe dosage measured mouse, rat and monkey is respectively 0.32,0.47,0.5 and 0.46mg/kg.PK is calculated and is based on Labeled dose.

The oral administration biaavailability of embodiment 15.

As shown in table 10, after the compound 1 that single dose is applied to mouse, rat and monkey, the oral bio of compound 1 Availability height (>80%).Bioavilability (being more than 100%) in monkey can be construed to 10 between IV and oral dose Times difference.Compound 1 is rapidly absorbed in all kinds, wherein observing that maximum systemic is dense in about 1-3 hours of administration Degree.

Table 10. is to mouse, rat, dog and the single mean serum pharmacokinetic parameter for orally administering rear compound 1 of monkey

^aExcipient：0.2%, MC/1%SLS

^bThe dispersion liquid of spray drying, excipient：2%TPGS/1.5%HPMCAS-HF/1.5%PVP-VA, contains 50mM Citrate pH5

^cThe dosage measured mouse, rat and monkey is respectively 0.95,0.68,2.6 and 4.4mg/kg.PK is calculated and is based on Labeled dose.

Table 11 provides the PI3K inhibitor described in International Patent Application Publication No. WO2011/087776 (" ' 776 application ") Pharmacokinetic data, the PI3K inhibitor each have and the identical core structural pharmacophore of compound 1.Respectively referring to 229th, 229, the compounds 705,709,735 and 772 of ' 776 applications on page 234 and 242.Compound 1 after intravenous delivery Systemic plasma clearance rate significantly (2.5 times -8 times) less than the result observed under study for action using other compounds.Blood plasma Clearance rate data and intravenous data, oral exposure, AUC and C_maxValue is consistent.These as shown by data compounds 1 and these chemical combination Thing, which is compared, has unexpected favourable pharmacokinetic properties.

Mean serum pharmacokinetic parameter of the compound 1 that table 11. is orally administered compared with control compounds

¹Intravenous push is studied, 0.5mg/kg labeled doses；Excipient 355PEG400/25%NMP/40% water

²Oral cavity gavage research, 3mg/kg labeled doses；Excipient 0.2%MC/1%SLS

Tissue distribution in the rat of embodiment 16.

Compound 1 is fully distributed into most tissues after 5mg/kg oral doses are applied to male rat.Referring to table 12. Brain and celiolymph (CSF) are to show the extremely low organ exposed to compound 1, wherein C_maxRespectively 111ng/g and 27ng/mL (<0.1% plasma exposure).Compound 1 is fully distributed in liver and kidney, wherein C_maxRespectively 11400 and 4770ng/g.Tissue Liver is followed with blood plasma ratio>Kidney>Heart>Lung>Spleen>Brain>CSF trend.Elimination pharmacokinetic of the compound 1 in each inspection organ Follow plasma kinetics.Evidence show single dose applies the tissue accumulation of rear compound 1.

The average tissue and plasma concentration and tissue of compound 1 and blood plasma ratio after the single 5mg/kg oral doses of table 12.

BQL=is less than measurable limit.By compound with the 2%TPGS/1.5% containing 50mM citrates pH5 The dispersion liquid administration of spray drying in HPMCAS-HF/1.5%PVP-VA.

Compound 1 in the mouse CIA model of embodiment 17.

By compound 1 with 2.5mg/kg BID (5mg/kg/ days), 5mg/kg BID (10mg/kg/ days) or 10mg/kg BID (20mg/kg/ days) is tested in arthritis (CIA) model that the collagen for the treatment of mouse induces.With 30mg/kg BID (60mg/kg/ days) orally give the Syk micromolecular inhibitors good fortune as reference standard he replace Buddhist nun.By compound in 12/12- It is administered 10 days in hour BID dosage regimens, until research terminates.Compound 1 is prepared with 0.2%MC, 1%SLS.Administered volume is 10mL/kg.2 after final dose, 4 and 12 hours collection end plasma samples.Gather all 4 pawls and two knees and process Handle for histopathology.

Treated using compound 1 and significant beneficial effect is shown in CIA models, as being commented by evaluating clinical arthritis Divide determined by the histopathology with joint.Compared with excipient control, with 2.5mg/kg BID compounds 1 (* d2-11), 5mg/kg BID compounds 1 (* d2-11), 10mg/kg BID compounds 1 (* d2-11) or 30mg/kg BID good fortune he replace Buddhist nun (* D2-11) arthritis score of the daily measure of the mouse for the treatment of significantly drops to normally.Referring to Fig. 1.When only considering registration When showing those pawl (treatment pawls) of clinical arthritis sign, 5mg/kg BID compounds 1 (* d5-11) or 10mg/kg are used The clinical arthritis scoring of the mouse of BID compounds 1 (* d2-11) treatment is remarkably decreased, but he replaces using 30mg/kg BID good fortune Buddhist nun treatment group does not have such case.Referring to Fig. 2.Those pawl (preventions of clinical arthritis sign are not shown when only considering registration Pawl) when, compared with excipient control group, use 2.5mg/kg BID compounds 1 (* d2,4-11), 5mg/kg compound 1BID (* D2-11), his clinic of mouse for being treated for Buddhist nun (* d5-9) of 10mg/kg compounds 1BID (* d2-11) or 30mg/kg BID good fortune Arthritis score is remarkably decreased.Referring to Fig. 3.

As shown in table 13, compared with excipient control group, with 30mg/kg BID good fortune, he replaces Buddhist nun's (25%), 2.5mg/kg The mouse of BID compounds 1 (28%), 5mg/kg BID compounds 1 (63%) or 10mg/kg BID compounds 1 (89%) treatment Be expressed as area under a curve (AUC) clinical arthritis scoring be remarkably decreased to normal.When only considering treatment pawl, use The arthritis score AUC of the mouse of 10mg/kg BID compounds 1 (79%) treatment significantly declines.When only considering prevention pawl, Compared with excipient control group, with 30mg/kg BID good fortune, he replaces Buddhist nun's (32%), 2.5mg/kg BID compounds 1 (42%), 5mg/ The arthritis score AUC of the mouse of kg BID compounds 1 (81%) and 10mg/kg BID compounds 1 (98%) treatment is notable Ground declines.

The clinical arthritis scoring of compound 1 in table 13.CIA models

*p<0.05ANOVA is examined with gram (this karr of Shandong)-watt (Li Si) Er Shi of Vehicle Control

Compound 1 in the mouse IBD models of embodiment 18.

The test PI3K gamma inhibitors compound 1 in the colitis model that CD40 is induced, to determine it to lysis Effect.By the way that anti-CD 40 monoclonal antibody (activator, i.e., activating antibodies are for neutralizing antibody) is injected into T and B cell defect Mouse (Rag 1-/- mouse) so as to induce systemic and intestines inflammation, cause by congenital immunity by way of colitis and consumption Property disease induces IBD CD40 models (see, e.g. Immunity 25, in August, 309-318,2006).In short, giving Rag1 knock-out mices IP injection anti-CD 40 monoclonal antibodies FGK45.Since the 0th day, with PBS, excipient or 5mg/kg B.i.d. or 10mg/kg b.i.d. compounds 1 treatment mouse.Compound 1 was applied with 10/14- hours b.i.d. dosage regimens IP With 7 days.Compound 1 is prepared with the 5%NMP/15%PEG-400/80% 0.5%HPMC-E50 aqueous solution.At the end of research, 2 hours collection serum and colonic specimen samples are analyzed for drug concentration after final dose.The daily measurement body weight during this research. At the end of research, the concentration of compound 1 in the plasma sample of collection in 2 hours after final dose is analyzed.Use efficient liquid phase Chromatography/tandem-mass spectrometry (HPLC/MS/MS) method determines concentration.

Injection anti-CD 40 monoclonal antibody simultaneously when starting within the 1st day there is body weight to subtract with the mouse of excipient or PBS treatments Gently (it is measured as the percentage change apart from baseline) and reaches peak value during at the 3-4 days, then recovers to baseline.As Fig. 4 Shown in, the weight loss that disease induces at the 3rd and 4 day was respectively 17.2% and 17% in PBS treatment groups, and excipient At the 3rd and 4 day it was respectively 12.8% and 12.6% in treatment group.Fig. 4 is also shown compared with excipient control group, uses 5mg/ kg b.i.d.(*p<0.05d3,**p<0.01d4) or 10mg/kg b.i.d. (* * * p<0.001d3-4) compound 1 is treated Weight loss is significantly inhibited in mouse.

Tissue concentration of the compound 1 of table 14 in the IBD models that CD40- is induced

Although above-mentioned is described in detail to a certain extent by example and embodiment for clearness of understanding Invention, it will be apparent that those skilled in the art can not depart from accompanying claims essence according to the teachings of the present invention Some changes and modification are carried out to the present invention in the case of god or scope.

Claims (7)

1. the compound of following formula：

Or its pharmaceutically acceptable salt.

2. pharmaceutical composition, includes compound according to claim 1 and pharmaceutically acceptable carrier or excipient.

3. the compound of claim 1 prepare be used to treat disease or illness selected from autoimmune disease or inflammatory disease or Mitigate the purposes in the medicine of its seriousness, the autoimmune disease or inflammatory disease are selected from asthma, atopic dermatitis, nose Inflammation, anaphylactia, chronic obstructive pulmonary disease, septic shock, idiopathic pulmonary fibrosis, apoplexy, burn, arthropathy, class wind Wet arthritis, systemic loupus erythematosus, atherosclerosis, acute pancreatitis, psoriasis, inflammatory bowel disease, ulcerative colitis Scorching, Crohn disease and Graves disease.

4. the purposes of claim 3, wherein the disease or illness are rheumatoid arthritis.

5. the compound of claim 1 is being prepared for suppressing the purposes in biological sample in the preparation of PI3K- γ kinase activities.

It is at least another more than suppression that 6. the compound of claim 1 is used for optionally suppression PI3K- γ isoforms in preparation Purposes in the preparation of PI3K isoforms.

7. the purposes of claim 6, another PI3K isoforms of wherein at least are selected from PI3K- α, PI3K- β, PI3K- δ and its group Close.